The present invention is directed to an effective diagnostic testing for the presence of gastric infection by the microorganism Helicobacter pylori. 
Helicobacter pylori is estimated to be responsible for up to 90% of the cases of peptic ulcer disease (PUD) afflicts over 10% of the US population sometime in their lifetime. Estimates for worldwide prevalence of H. pylori infections range from 300 million to over two-thirds of the world's population. H. pylori infection is also associated with 650,000 annual cancer deaths worldwide from gastric adenocarcinoma. The US Communicable Disease Center recommends testing all patients presenting with PUD for diagnosis of H. pylori. 
At present there are no test methods for H. pylori that satisfy the ideal conditions of being non-invasive, rapid, easy to administer, have low capital equipment and per-patient test costs, and capable of being conducted in entirety during a clinician's office visit. Currently practiced approaches for H. pylori testing can be broken down into invasive (endoscopy required) and non-invasive procedures. Examples of non-invasive tests include: determination of antibodies to H. pylori in blood, serum, or saliva; detection of H. pylori antigens in stool samples; and functional tests for the presence of the bacterium's urease enzyme with isotope-labeled urea breath tests (UBT).
Although the non-invasive antibody-based tests are relatively easy to perform, they have not proved to be reliable in the general practitioner's office. Furthermore, they incur a blood draw and the costs associated with the blood draw procedure. Additionally, antibody tests cannot provide a test-of-cure to demonstrate successful antibiotic treatment.
A variety of diagnostic procedures have evolved based on functional tests establishing presence of the urease enzyme produced by H. pylori. Urease, an enzyme found at high concentrations in the duodenum of infected individuals, hydrolyzes urea to ammonia (NH3) and carbon dioxide (CO2). Tests for gastric urease, vide infra H. pylori, rely on measures of the hydrolytic by-products of urea. With respect to non-invasive diagnosis, breath-based tests for expired isotopically-labeled CO2 liberated from ingested isotopic urea are well known in the literature. Graham described a breath test for Camphylobacter (Helicobacter) based on measurement of 13CO2 released after hydrolysis of ingested 13C-labeled urea (Graham, Lancet, May 23, 1987, p1174–1177). Others have used the rapid production of isotopically labeled CO2 from ingested 14C-urea or 13C-urea to determine the presence or absence of the organism.
Measurement of ammonia production by the hydrolytic activity of urease is the basis for the invasive CLO and rapid urease tests. However, to execute these tests, invasive endoscopy procedures are required. They are therefore neither simple, economical, convenient for the patient, or executable in general clinical practices.
For a number of reasons, it has proven difficult to make measurements of breath ammonia liberated as a by-product of H. pylori urease. First, ammonia exists primarily as the ammonium ion at the physiologic pH of blood, and at the pH of gastric juice there is essentially no free ammonia. While ammonia readily crosses the stomach and alveolar lining, ammonium ions are not readily absorbed. Therefore very little ammonia finds its way from the stomach, traffics through the circulatory system, and passes into expired air, consequently making it difficult to measure.
A second major reason is the tight regulation of ammonia and ammonium levels by the liver and kidneys. Prior to general circulation, blood from the gut is circulated through the hepatic portal vein to the liver. Normally, the combination of periportal urea cycle enzymes and perivenous glutamine synthetase results in almost complete removal of NH3 from blood flowing through the portal vein. Furthermore, at typical blood pH levels of 7.4, ammonia that does pass into the general circulation will exist primarily as ammonium ions that are removed by the kidney. This homeostatic regulatory system is therefore expected to minimize any fluctuations in circulating ammonia. Consequently, only minimal variations in breath ammonia would be expected either in normal individuals or, by comparison, individuals infected with H. pylori. 
The literature corroborates the difficulty measuring ammonia directly in breath and lack of clinical evidence differentiating H. pylori individuals based on breath measures, such that a simple diagnosis via breath analysis is not expected. Lipski (Lipsky PS et al., Aust NZ J Med 22:311,1992) and Plevris (Plevris JN et.al., Lancet:1104) found no difference in blood ammonia concentration between H. pylori positive and negative patients. Only by looking at 15NH4+ excretion in urine was Jicong (Jicong W et al., J. Clin Micro. 30(1):181–4, 1992) able to demonstrate a difference between pylori positive and negative subjects using nitrogen based assays. U.S. Pat. No. 4,947,861 suggests that by absorbing the water vapor from the breath prior to collecting a test sample, breath ammonia might be measured. However, he offers no evidence to demonstrate the utility of this maneuver and further, offers no teaching of its clinical utility or basis for deriving diagnoses. Similarly, Katzman (U.S. Pat. No. 6,067,989) suggests the use of near infrared analyzer for measuring breath changes in by-products (CO2 & NH3) of hydrolyzed isotopically labeled urea. Again however, Katzman's method does not teach diagnosis via ammonia, offering support only for measuring the 13C-labeled CO2 by-product as measured by others (Graham DY et.al., Lancet, 1174–77, May 23, 1987).
Isotopic labeling has been critical in other breath measurement diagnostics for several reasons. Labeling provides advantage towards sensitive and specific distinction of the labeled reporter by-product using sophisticated instrumentation. The specific measurement of the label enables these assays to distinguish and quantify the urea hydrolysis product(s) in the presence of unlabled native hydrolysis products. For instance, as in the case of isotopic CO2 based H. pylori breath testing, the use of 14C-labeled urea allows specific detection of the 14CO2 urea byproduct at nanomolar concentrations despite millimolar CO2 concentrations in the basal breath.
With respect to use of labeled urea, it is important to appreciate that the hydrolytic by-products of CO2 and NH3 generated within the gastrointestinal tract have vastly different fates within the body. As indicated, ammonia is tightly controlled by homeostatic mechanisms regulating physiological processing and circulating levels with little or no role for clearance by exhalation. In contrast, CO2 has markedly different regulatory processes affecting its circulatory concentration with its major route for clearance occurring through the lungs. Therefore, despite labeled CO2 being measurable in breath and serving diagnostically via the UBT method, it is not to be expected that ammonia would provide a parallel alternative avenue to diagnosis, much less be manifest in any diagnostically useful pattern in the breath.
There is a need for a simple, rapid non-invasive diagnostic test for H. pylori, based on measuring ammonia in breath, without the use of isotopically labeled reagent.
These and other limitations and problems of the past are solved by the present invention.